New monoclonal antibody for the selective expansion of NKG2c+ NKG2a- adaptive NK lymphocytes and method thereof

Abstract

The present invention relates to a novel monoclonal antibody and its use in a specific method for the expansion of adaptive NK lymphocytes expressing the activating receptor CD94 / NKG2C. In particular, the invention relates to a monoclonal antibody designed for the selective expansion of adaptive NK lymphocytes NKG2C+CD57+NKG2A- selfKIR+ FcϵRy- / low and Syk- / Iow, characterized by the absence of the inhibitory receptor PD-1. The invention also describes a specific method for the expansion and selection of this particular cell population, as well as the use of the aforementioned population in the medical field.

Description

New monoclonal antibody for the selective expansion of NKG2C+ NKG2A- adaptive NK lymphocytes and method thereof.Applicant : Universita degli Studi di GenovaInventors : Prof, ssa Simona Sivori, Prof, ssa Mariella Della Chiesa : Dott.ssa Chiara Giordano, Prof.ssa Simona Carlomagno, Dott.ssa Chiara SettiTechnical Field

[0001] The present invention relates to a novel monoclonal antibody and its use in a method for expanding adaptive NK lymphocytes expressing the activating receptor CD94 / NKG2C.State of the art

[0002] Natural Killer (NK) lymphocytes are cytotoxic cells belonging to the innate immunity, capable of killing tumor cells or cells infected by viruses by providing effective responses faster than T lymphocytes. Their activity is finely regulated by a series of activating and inhibitory receptors [1], The latter include a panel of receptors that recognize HLA class I molecules (HLA- I), i.e. the CD94 / NKG2A heterodimer and the inhibitory KIR (Killer-cell Immunoglobulin-like Receptors) receptors [2], In particular, the CD94 / NKG2A heterodimer is conserved and recognizes the non-classical molecule HLA-E, while KIR are highly polymorphic and recognize epitopes of classical HLA-I molecules (-A, -B to -C). Specifically, KIR2DL1 recognizes HLA-C alleles with the C2 epitope, KIR2DL2 / L3 recognizes HLA-C alleles with the Cl epitope, and KIR3DL1 recognizes HLA-A and HLA-B alleles characterized by the Bw4 epitope [3], Furthermore, NK cells can express activating forms of HLA-l-specific receptors that include the CD94 / NKG2C heterodimer (counterpart of CD94 / NKG2A, fig. 1), KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, and KIR3DS1. These forms often recognize the same ligands as the inhibitory receptor but with different affinities [4, 5], The recognition of self HLA-I molecules by CD94 / NKG2A and KIR inhibitors is a fundamental requirement for the maturation of NK cellsso that, through a process called "education", NK cells are generated that are functionally competent towards tumor cells and virus-infected cells, but tolerant towards self [6],

[0003] In response to a common viral infection, caused by human cytomegalovirus (CMV), a particular population of NK cells, called "memory-like" or adaptive, can be generated in both healthy and pathological individuals [7-10], This population of NK cells is characterized by the expression of the activating receptor CD94 / NKG2C, by strong antiviral and antitumor effector functions (especially after antibody-mediated activation) and by longevity, an unusual feature for cells belonging to the innate immunity

[0011] , Adaptive NKG2C+NK cells are characterized by a highly differentiated profile, CD56dimCD16+CD57+NKG2A- self KI R+and by an altered expression of transcription factors and adaptor proteins involved in signal transduction, following epigenetic modifications. Among these characteristics of adaptive NK lymphocytes, the decreased expression of the adaptor molecule FcsRy is the most common in CMV seropositive (CMV+) individuals. This attribute is associated with a greater cytotoxic capacity of adaptive NK lymphocytes against opsonized cells, i.e. cells coated with immunoglobulin G (IgG), through the engagement of CD16, the receptor for the Fc portion of IgG, by a mechanism known as ADCC (antibody-dependent cellular cytotoxicity). This greater ADCC capacity of FcsRy- adaptive NK cells is determined by a more potent transduction of the signal induced via CD16. In fact, in the absence of FcsRy, CD16 is coupled only to the adaptor protein CD3^ which is equipped with three ITAM (Immunoreceptor Tyrosine-based Activation Motif) sequences and can therefore transduce a stronger signal than FceRy which has only one ITAM [12-15], It is interesting to consider how adaptive NK lymphocytes, although generated following a viral infection, can also play an important role against tumor pathologies as suggested by various studies which have highlighted their protective effects against relapses of acute leukemia [16-19],

[0004] In light of these peculiar characteristics, the population of adaptive NK lymphocytes NKG2C+has a high potential in the field of cellular immunotherapies against both infectious diseases and different types of tumors, including haematological malignancies, such as acute leukemias and lymphomas, and solid tumors, such as head and neck tumors, ovarian carcinoma or colorectal carcinoma. In fact, some clinical trials based on the use of adaptive NK lymphocytes are currently underway in patients affected by acute myeloid leukemia(NCT03081780) or ovarian and peritoneal carcinoma (NCT03213964) or other solid tumors (gastric, colorectal, breast, head and neck) (NCT03319459).

[0005] Current approaches to generate NK cells for immunotherapies require expansion platforms to obtain large numbers of cells. There is therefore a need for expansion methods able to effectively stimulate the proliferation of adaptive NK lymphocytes that will subsequently be used for adoptive transfer in selected patients, alone or in combination with novel biological tools (e.g. so-called cellular engagers) to maximize their reactivity against tumors or virus-infected cells [20, 21].

[0006] The methods described so far to expand NKG2C+adaptive NK cells, although showing good expansion efficiency, show some disadvantages that push to design new methods to overcome these difficulties. Patent WO2014037422A1 and a recent scientific publication

[0022] describe a protocol for the ex vivo expansion of NKG2C+NK cells from selected healthy donors, which is based on a co-culture system with cell lines transfected to express in their membrane the ligand recognized by CD94 / NKG2C (i.e. the non-classical HLA class I molecule HLA-E). This co-culture method may however raise concerns regarding the in vivo use of expanded NK cells in the presence of genetically manipulated cell lines. Indeed, the use of genetically modified cell lines is associated with several regulatory issues that require further rigorous and expensive analyses, especially considering large-scale use (https: / / health.ec.europa.eu / medicinal-products / eudralex / eudralex-volume-4_en; https: / / www.ema.europa.eu / en / human-regulatory / research- development / compliance / good-manufacturing-practice). Patent EP3539552A1 describes a method based on the use of a specific peptide that mimics CMV-derived peptides to induce the activation and expansion of adaptive NKG2C+NK cells in vivo and ex vivo. However, both the patent and the accompanying study

[0023] indicate the need to employ a combination of pro-inflammatory cytokines (IL-12, IL-18 and IL-15) to achieve the activation and expansion of adaptive NK lymphocytes in response to the specific peptides used. Prolonged culture in the presence of pro-inflammatory cytokines can induce the co-expression of the inhibitory receptor CD94 / NKG2A on NKG2C+NK cells, reducing the effector function of adaptive NK cells and their potential in immunotherapies

[0023] , Furthermore, as indicated in EP3539552A1, this method can also induce the expression of other inhibitory receptors such as PD-1 and LAG-3 further compromising the functionality of adaptive NK cells.

[0007] There remains a need for a method to expand adaptive NK lymphocytes that does not require the use of genetically modified cell lines or peptides or combinations of pro- inflammatory cytokines.

[0008] The present invention therefore provides a flexible method, technically and economically more advantageous, and therefore more suitable to the needs of large-scale production.Summary

[0009] It is an object of the present invention is a monoclonal antibody isolated against human CD94 or an antigen-binding fragment thereof comprising three regions determining the complementarity of the heavy chain, HCDR1, HCDR2, HCDR3 and three regions determining the complementarity of the light chain LCDR1, LCDR2, LCDR3. Specifically, in an embodiment according to the invention a) the amino acid sequence of HCDR1 is the amino acid sequence reported in SEQ ID NO:20; b) the amino acid sequence of HCDR2 is the amino acid sequence reported in SEQ ID NO: 21; c) the amino acid sequence of HCDR3 is the amino acid sequence reported in SEQ ID NO: 22; d) the amino acid sequence of LCDR1 is the amino acid sequence reported in SEQ ID NO: 9; e) the amino acid sequence of LCDR2 is the amino acid sequence YTS f) the amino acid sequence of LCDR3 is the amino acid sequence reported in SEQ ID NQ:10

[0010] It is another object of the present invention an isolated monoclonal antibody of the IgGl class or an antigen-binding fragment thereof comprising a variable domain of the light chain of SEQ ID NO: 8 and a variable domain of the heavy chain of SEQ ID NO: 19, wherein said variable domain of the light chain comprises an LCDR1 light chain complementarity determining region, an LCDR2 light chain complementarity determining region and an LCDR3 light chain complementarity determining region and wherein said variable domain of theheavy chain comprises an HCDR1 heavy chain complementarity determining region, an HCDR2 heavy chain complementarity determining region and an HCDR3 heavy chain complementarity determining region; these regions have been determined with IMGT.

[0011] In one embodiment the LCDR1 region of the antibody comprises amino acids 9 through 14 of SEQ ID NO: 8, the LCDR2 region of the antibody comprises amino acids 32 through 34 of SEQ ID NO: 8, and the LCDR3 region of the antibody comprises amino acids 71 through 79 of SEQ ID NO: 8.

[0012] In one embodiment the HCDR1 region of the antibody comprises amino acids 8 through 15 of SEQ ID NO: 19 the HCDR2 region of the antibody comprises amino acids 33 through 40 of SEQ ID NO: 19, and the HCDR3 region comprises amino acids 79 through 89 of SEQ ID NO: 19.

[0013] It is a further object of the present invention an isolated nucleic acid for the production of the anti-human CD94 antibody, in particular an isolated nucleic acid comprising the sequences of SEQ ID NO. 1 and 11 is preferred.

[0014] The monoclonal antibody according to the invention or a fragment thereof is particularly suitable for use in medicine.

[0015] A further object of the present invention is also an in vitro method for selecting and expanding adaptive NK lymphocytes NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / Iowcomprising the following basic steps: i. Isolating a population of peripheral blood mononuclear cells from a biological sample of a subject with CMV+serology, ii. Performing a depletion of T lymphocytes to obtain a population enriched with NK lymphocytes, iii. Cultivating the population obtained at point ii. in a culture medium comprising the antibody according to the present invention and at least one of the cytokines IL-2 or IL-15 for a time between 10 and 14 days,

[0016] In a preferred embodiment said method allows to obtain an expanded population of NK cells enriched with NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / Iowcells further characterized by the absence of the inhibitory receptor PD-1.

[0017] With the method according to the invention it is also possible to select NK cells expressing KIRs specific for HLA-C1 or for HLA-C2 to produce an expanded population of NK cells enriched with NKG2C+CD57+NKG2A-selfKIR2DL2-L3+Fc£Ry- / lowand Sylc / Iowor NKG2C+CD57+NKG2A“selfKIR2DLl+Fc£Ry- / lowand Sylc / Iowcells.

[0018] It is an additional object of the present invention a population of adaptive NK lymphocytes NKG2C+CD57+NKG2A“selfKIR+Fc£Ry_ / low andSyk_ / Iowor a composition comprising adaptive NK lymphocytes NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / Iowdirectly obtainable with the method according to the invention. In a preferred embodiment, said population is further characterized by the absence of the inhibitory receptor PD-1.

[0019] This selected population is highly effective in the treatment of haematological diseases, solid tumors, immunodeficiencies.

[0020] Further objects of the invention will be evident from the detailed description and the examples that follow.Brief description of the figures

[0021] FIGURE 1 Schematic representation of CD94 / NKG2A and CD94 / NKG2C heterodimers

[0022] On the left is the inhibitory CD94 / NKG2A heterodimer where the presence of two ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) sequences in the intracytoplasmic portion of the NKG2A molecule is highlighted. Following receptor engagement, ITIMs transduce a signal capable of turning off the main cellular functions of NK lymphocytes (e.g. cytotoxicity, cytokine release, proliferation). On the right is the activating CD94 / NKG2C heterodimer. In this receptor, the intracytoplasmic portion of the NKG2C polypeptide chain is shorter than that of NKG2A and does not have ITIMs, but the intramembranous portion allows it to couple with the signal transducing molecule DAP-12, which instead contains the ITAM (Immunoreceptor Tyrosine-based Activation Motif) sequences. Following receptor engagement, the ITAMs of DAP-12 transduce a signal capable of activating the cellular functions indicated above. The CD94 polypeptide chain does not have sequences capable of activating or deactivating cellular functions and binds covalently to the NKG2A or NKG2C molecules. The figure was created with BioRender.

[0023] FIGURE 2 NKG2C+CD57+ / _adaptive NK lymphocytes from healthy CMV+donorsa) Selection strategy of adaptive NK lymphocytes isolated from peripheral blood of healthy CMV+donors. This strategy was used to identify NK cells both on total mononuclear cells and on mononuclear cells post-depletion of CD3+T lymphocytes at day zero of culture. The lymphocyte population was identified thanks to its physical characteristics, from which any cell doublets were then excluded. The NK cells were then selected as CD56+CD3-, CD19-, CD14- Fixable Viability Stain- (marker that identifies non-viable cells). NK cells were then analyzed for the expression of CD16, NKG2C, CD57 and NKG2A: in particular, the adaptive NKG2C+cells were divided into the NKG2C+CD57- and NKG2C+CD57+subpopulations, on which the analyses of this study focused. The percentage of the different NK subcategories is indicated in the corresponding quadrant of each graph. Where necessary, the monocytes identified in the first graph FCS and SSC were also evaluated. b) Selection strategy of adaptive NK lymphocytes following culture and expansion induced by monoclonal antibody with IL-2 or IL-15. The selection method is similar to that used on day zero, but the selection on physical parameters was adapted to the larger size of the stimulated lymphocytes. NK lymphocytes, identified as CD56+CD3- CD19-CD14- Fixable Viability Stain- cells, essentially represent the only detectable post-culture population. Adaptive NK cells NKG2C+CD57- and NKG2C+CD57+were then examined for the expression of specific markers and for their functional capacities, as described below. c) Selection strategy of NK lymphocytes expressing a single KIR (self-KIR) and lacking NKG2A and other KIR receptors. After selecting NK cells as in panels a-b, the frequency of NK lymphocytes expressing as the only HLA-l-specific inhibitory receptor KIR2DL1 (recognizes HLA-C2) was measured by combining anti-KIR2DLl-PE with a mixture of anti-KIR2DL2-L3-APC, anti-KIR3DLl-APC and anti-NKG2A-APC antibodies. The frequency of NK lymphocytes expressing as the only HLA-I specific inhibitory receptor KIR2DL2-L3 (recognizes HLA-C1) was measured by combining anti-KIR2DL2-L3-PE with a mixture of anti-KIR2DLl-APC, anti-KIR3DLl-APC and anti-NKG2A-APC antibodies. d) Frequency of NKG2C+CD56dimNK lymphocytes identified according to the panel a strategy, in the peripheral blood of healthy CMV+(n=60, filled black circles) and CMV- (n=38, empty black circles) individuals. For CMV+donors, the frequencies of the twoNK subpopulations NKG2C+CD57+(filled black triangles) and NKG2C+CD57“ (filled black squares) are also shown. The mean frequency of each subgroup with its standard deviation is shown in the graph.

[0024] FIGURE 3 Schematic representation of the expansion protocol

[0025] The figure shows the steps of the expansion procedure in chronological order. The figure was created with BioRender.

[0026] FIGURE 4 Frequency of NK, B, T lymphocytes and monocytes at different culture times in the presence of anti-CD94 mAb and IL-2 or IL-15

[0027] The relative frequency of the various cell types present in culture is reported at the various times analyzed starting from day 0 wherein the mononuclear cells, post-depletion of T lymphocytes, are cultured in the presence of the anti-CD94 mAb and IL-2 (left panel) or IL- 15 (right panel), up to day 14. The percentage of the different cell populations was evaluated by multiparametric flow cytometric analysis using the following panel of monoclonal antibodies: anti-CD56-PC7, anti-CD3-PerCPcy5.5, anti-CD19-VioBlue, anti-CD14-APC. Non- viable cells were excluded from the analysis using the Fixable Viability Stain 510 marker. The frequencies reported at the various time points represent the mean value of n=13 experiments with different donors. The bars indicate the standard deviation on the mean frequency of each cell population.

[0028] FIGURE 5 Expansion of adaptive NKG2C+NK lymphocytes induced by anti-CD94 mAb

[0029] The number of NKG2C+NK lymphocytes present in culture was assessed at various culture times starting from day 0 when 105mononuclear cells per well (96-well plates) were cultured as indicated in figure 3 (CD3-depleted PBMC). Cultures were performed with (square symbol) or without (circle symbol) the addition of mAb, in the presence of IL-2 (left panel) or IL-15 (right panel). The number of NKG2C+NK lymphocytes was calculated for each experiment (n=13) based on the composition of the cell culture as indicated in figure 3. In particular, the frequency of NKG2C+NK lymphocytes was analyzed at each time point by flow cytometry as indicated in the selection strategy in figure 2a-b. The bars indicate the standard deviation of the mean value. For each time point analyzed (7, 10, 14 days), the number of cells in the culture in the presence of mAb was compared with the number in the culture in theabsence of mAb using the Mann-Whitney test. Statistical significance (p value) is reported at each point (*p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001).

[0030] FIGURE 6 Progressive proliferation of adaptive NK lymphocytes NKG2C+CD57+ / _induced by anti-CD94 mAb.

[0031] In each experiment, a portion of the post-depletion mononuclear cells obtained from the various CMV+ donors was labeled with CFSE and cultured according to the protocol in figure 3 in the presence or absence of mAb. In a) a representative experiment of n=13 experiments performed, starting from day 0, is shown, wherein the CFSE-labeled NK lymphocytes were analyzed as indicated in the strategy in figure la-b.

[0032] In b) and c) the proliferation of NKG2C+NK lymphocytes induced by cytokine+mAb, demonstrated by the dilution of CFSE, is shown at various times (7, 10, 14 days) in parallel with the proliferation in the presence of cytokine but in the absence of mAb. In the right panels the reciprocal expression of NKG2C and CD57 on NK lymphocytes present in culture was analyzed as described in fig. 2a-b. The frequency of NKG2C / CD57 NK cells is indicated in each quadrant and shows the progressive increase of NKG2C+CD57+NK cells in the presence of both IL-2 (b) and IL-15 (c) mAbs.

[0033] FIGURE 7 Increased frequency of NKG2C+, NKG2C+CD57+and NKG2C+CD57“ adaptive

[0034] The frequency of the different NK populations present on day 0 (circular symbol) and day 14 of culture with IL2+mAb (square symbol) or IL-15+mAb (triangular symbol) was assessed by flow cytometric analysis according to the strategy in fig. 2a-b. The frequency of total NKG2C+NK lymphocytes is reported in the left panel, the frequency of NKG2C+CD57+NK lymphocytes in the central panel, while the frequency of NKG2C+CD57“ NK lymphocytes is shown on the right for n=13 experiments performed. The bars indicate the standard deviation of the mean value. The frequency of the different NK populations on day 0 compared to day 14 in IL-2 or IL-15 was analyzed by the Mann-Whitney test. Statistical significance is reported in the various panels (*p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001).

[0035] FIGURE 8 Expansion of adaptive NK lymphocytes NKG2A-, expressing a single self-KIR, induced by anti-CD94 mAb

[0036] The frequency of the various NK populations present on day 0 (circular symbol) and on day 14 of culture with IL-2+mAb (square symbol) or IL-15+mAb (triangular symbol) was analyzed by flow cytometric analysis according to the strategies in fig.2a-c.

[0037] In a) the frequency of NKG2A+NK lymphocytes (n=13) is reported, in b) the frequency of NKG2A+NKG2C+NK lymphocytes (n=13), in c) the frequency of NK lymphocytes expressing exclusively a single self-specific KIR (KIR2DL1 in n=2, KIR2DL2-L3 in n=6) and lacking NKG2A and the other KIR receptors. The frequencies of NK lymphocytes expressing exclusively a single self-specific KIR relative to the NKG2C+CD57+and NKG2C+CD57“ populations are reported in panels d) and e), respectively.

[0038] The bars indicate the standard deviation of the mean value. The frequency of the various NK populations at day 0 versus day 14 in IL-2 or IL-15 was compared using the Mann- Whitney test. Statistical significance is reported in the various panels (*p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001).

[0039] FIGURE 9 Phenotypic characterization of NKG2C+CD57+adaptive NK lymphocytes pre- and post-expansion induced by anti-CD94 mAb

[0040] In a) the expression of adaptor molecules involved in the transduction of activating signals FceRy, Syk and CD3^ was analyzed on the population of adaptive NK lymphocytes NKG2C+CD57+pre-expansion (day 0) and post-expansion (day 14) with IL-2+mAb or IL- 15+mAb, by intracellular cytofluorimetric analysis. In parallel, the expression of the activating receptor CD16, which transduces signals through the above-mentioned molecules, was evaluated on the same populations. The frequency of expression of the various markers on NKG2C+CD57+cells is indicated by the grey intensity of the Heatmap. The frequency of the different markers on NK lymphocytes NKG2C+CD57+on day 0 compared to day 14 in IL-2 or IL- 15 was compared by the Mann-Whitney test. No statistically significant difference was observed. For brevity, NK lymphocytes cultured with cytokines alone have been omitted from the Heatmap, as no significant differences were observed either at day 0 or day 14.

[0041] In b) the frequency of expression of inhibitory receptors representing critical immune checkpoints, PD-1, TIM-3, LAG-3, TIGIT, is reported in a way similar to a) on the population of adaptive NK lymphocytes NKG2C+CD57+pre-expansion (day 0) and post-expansion (day 14) with IL-2 or IL-2+mAb alone and with IL-15 or IL-15+mAb alone. The frequency of the variousmarkers on day 0 compared to day 14 in IL-2- or IL-15 with and without mAb was compared using the Mann-Whitney test (*p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001). Statistical significance reported in the panels refers to the comparison of the different conditions at day 14 with day 0. No statistically significant difference was observed when IL-2 vs. IL-2+mAb and IL-15 vs. IL-15+mAb conditions were compared.

[0042] FIGURE 10 Expanded NKG2C+CD57+adaptive NK lymphocytes are highly functional and have high ADCC capabilities

[0043] The degranulation capabilities of NKG2C+CD57+adaptive NK lymphocytes isolated from a healthy CMV+donor after 14 days of expansion in the presence of IL-2 or IL-15 and anti-CD94 mAb were examined in n=6 experiments with different donors. NKG2C+CD57+adaptive NK cells were identified according to the selection strategy in fig. 2b.

[0044] Surface expression of CD107a was assessed on NKG2C+CD57+NK cells after 3 hours of co-culture at an effector (expanded NK) / target ratio of 1 / 1 with the indicated tumor cell lines, i.e. K562, LCL 721.221 wild type (indicated as 221wt), 721.221AEH (i.e. transfected to express HLA-E, indicated as 221E), RAJI, rituximab-opsonized RAJI (indicated as RAJI+Rtx) or with the medium alone as a negative control (indicated as NK). The 221E tumor cell line allows to verify the ability of NKG2C+NK cells to recognize their ligand (HLA-E), while the Rtx-opsonized RAJI line allows to evaluate the ADCC capabilities via CD16.In a) the aggregated data of the various experiments are reported, wherein the bars indicate the standard deviation of the mean value. The different degranulation capacity of adaptive NK lymphocytes was compared between NK lymphocytes alone versus NK+K562, between NK+221wt versus NK+221E and between NK+RAJI versus NK+RAJI+Rtx using the Wilcoxon test (rank test). Statistical significance is reported for each pair of variables examined (*p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001).In b) a representative experiment related to NKG2C+CD57+NK lymphocytes expanded in IL- 2+mAb is shown. The percentage of NKG2C+CD57+NK lymphocytes expressing CD107a is indicated in the upper right quadrants in the different experimental conditions.Description of the invention

[0045] The present invention relates to a new IgGl class monoclonal antibody directed against the CD94 receptor. The monoclonal antibody according to the invention, thanks to itsbinding properties, is in particular also able to bind to the CD94 / NKG2C activating receptor of adaptive NK lymphocytes.

[0046] The present invention also relates to an alternative in vitro method for expanding adaptive NK lymphocytes by using said monoclonal antibody, and in particular a method for expanding a subpopulation of NK lymphocytes expressing the CD94 / NKG2C activating receptor, called adaptive or "memory" NK lymphocytes, endowed with specialized effector functions and an unusual longevity. The method according to the invention provides that said population of adaptive NK cells is isolated from samples obtained from donors with selected positive serology to CMV and is specifically aimed at obtaining a CD56+CD16+NKG2C+CD57+NKG2A“self KI R+population.

[0047] With the method according to the invention it is therefore also possible to selectively expand cells with a given KIR specificity. In a preferred embodiment it is possible to select a population of "educated" cells expressing KIR specific for HLA-C1 (KIR2DL2-L3) or for HLA-C2 (KIR2DL1).

[0048] The selected population is characterized by a high immunotherapeutic potential and, following expansion, can be used in cellular immunotherapies aimed at improving and redirecting the cytotoxic activity of NK cells against specific biological targets (e.g. tumor antigens), also through innovative methods such as engineering with CAR (Chimeric Antigen Receptors).

[0049] With the method and the antibody according to the invention, it is in particular possible to obtain a population CD56+CD16+NKG2C+CD57+NKG2A“self KI R+FcERy- / lowSylc / Iow, i.e. characterized by a complete adaptive phenotype, associated with high ADCC capabilities.

[0050] The method according to the invention presented here can be used to select cells suitable for use as a non-limiting example in the therapy of various pathologies, in particular leukemias, tumors and immunodeficiencies.

[0051] The selected and enriched cells can then be used for medical purposes and transferred to the patient.

[0052] In one embodiment, the invention relates to a cell population obtainable with such a method, or to a composition comprising such a cell population for use in medicine, inparticular for use in the treatment of pathologies that respond to therapy with NK cells CD56+CD16+NKG2C+CD57+NKG2A“self KI R+.

[0053] Such pathologies are represented, for instance, by immunodeficiencies, by haematological pathologies, including tumours, and by solid tumours; by way of non-limiting example, acute myeloid and lymphoid leukemias, lymphomas, myeloma, ovarian, peritoneal, gastric, colorectal, breast, head and neck, pancreatic carcinoma, sarcomas and brain tumours.

[0054] The method according to the invention is based on the use of a new antibody, which interacts with high specificity with the CD94 molecule, allowing the selection from samples isolated from subjects with positive CMV serology of adaptive NK cells that express in particularthe activating heterodimer CD94 / NKG2C, but which lack the inhibitory heterodimer CD94 / NKG2A. This latter receptor represents a critical inhibitory checkpoint in negatively regulating the cytotoxic activity of NK cells.

[0055] The invention therefore also relates to said antibody and to compositions that comprise the antibody and / or fragments thereof.

[0056] The antibody according to the present invention is a monoclonal antibody or a fragment thereof characterized by binding specifically to CD94, binding specifically to the human CD94 / NKG2C heterodimer and promoting the selection of NKG2C+CD57+NKG2A“ self KIR+cells.

[0057] The antibody according to the invention was obtained from murine hybridoma generated using splenocytes obtained from immunocompetent mice (BALB / c strain), immunized with human NK cells and fusing them in vitro, thanks to the use of polyethylene glycol, with cells of a particular non-antibody-secreting murine myeloma line called P3U1, in order to obtain immortalized hybridomas

[0024] , Different antibodies were obtained, and, after tests, the antibody according to the present invention was selected.

[0058] The variable regions of the antibody that include the CDR portions (Complementarity Determining Regions) capable of interacting with the antigen, i.e. the CD94 molecule that forms the CD94 / NKG2x complexes, were sequenced.

[0059] The antibody according to the invention is therefore a murine IgGl monoclonal antibody characterized by the following sequences:Nucleotide sequence of the light chain (kappa) of Ig - SEQ ID NO.lGTCACCATCA GTTGCAGGGC AAGTCAGGAC ATTGACAATT CTTTAAACTG GTATCAGCAGAAACCAGATG GAACTGTTAA ACTCCTGATC TCCTACACAT CAAGATTACA CTCAGGAGTCCCATCAAGGT TCAGTGGCAG TGGGTCTGGA ACAGATTATT CTCTCACCAT TACCAACCTGGAGCAAGAAG ATATTGCCAC TTACTTTTGC CAACAGGGTA ATACGCTTCC GTACACGTTCGGAGGGGGGA CCAAGCTGTable 1Table 1 lists the numbers of nucleotides that delimit the framework regions (FR1-IMGT, FR2- IMGT, FR3-IMGT, FR4-IMGT) and the complementarity determining regions (LCDR1-IMGT, LCDR2-IMGT, LCDR3-IMGT) of the Ig light chain (kappa) (SEQ ID NO.l), together with their respective nucleotide sequences. The corresponding amino acid sequence is shown below with the CDR sequences highlighted.Amino acid sequence of the light chain (kappa) of Ig - SEQ ID NO:8VTISCRASQDIDNSLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTITNLEQEDIATYFCQQGNTLPYTFGGGTKLLCDR1=QDIDNS of SEQ ID NO:9LCDR2=YTS Not included in sequence listing (<4 aa)LCDR3=QQGNTLPYT of SEQ ID NQ:10Nucleotide sequence of Ig heavy chain - SEQ ID NO:11AAGTTGTCCT GCACAGCTTC TGGCTTCAAC ATTAAAGACA CCTATATGCA CTGGGTGAAGCAGAGGCCTG AACAGGGCCT GGAATGGATT GGAAATATTG ATCCTGCGAA TGCTAATACTAAATGTGCTT CGAAGTTCCA GGGCAAGGCC ACTATAACAG CAGACACATC CTCCAACACAGCCTACCTGC AGCTCAGCAG CCTGACATCT GAGGACACTG CCGTCTATTA CTGTGCTGGAGGGAGGGGTC TGGCCTGGTT TGCTTACTGG GGCCAAGGGA CTCTGGTCAC TGTCTCTGCA 1Table 2

[0060] Table 2 lists the numbers of nucleotides that delimit the framework regions (FR1H- IMGT, FR2H-IMGT, FR3H-IMGT, FR4H-IMGT) and the complementarity determining regions (HCDR1-IMGT, HCDR2-IMGT, HCDR3-IMGT) of the Ig heavy chain (SEQ ID NO.ll), together with their respective nucleotide sequences. The corresponding amino acid sequence is shown below with the CDR sequences highlighted.Amino acid sequence of the Ig heavy chain -SEQ ID NO: 19KLSCTASGFNIKDTYM H WVKQRPEQG LEWIG NIDPANANTKCASKFQG KATITADTSSNTAYLQLSSLTS E DTAVYYC AG G RG LAW F AY WGQGTLVTVS ACDR1=GFNIKDTY - SEQ ID NO:20HCDR2=IDPANANT - SEQ ID NO:21HCDR3=AGGRGLAWFAY - SEQ ID NO:22The present invention therefore concerns a monoclonal antibody isolated against human CD94 or an antigen-binding fragment thereof comprising three regions determining the complementarity of the heavy chain, HCDR1, HCDR2, HCDR3 and three regions determining the complementarity of the light chain LCDR1, LCDR2, LCDR3.

[0061] Specifically in an embodiment according to the invention a) the amino acid sequence of HCDR1 is the amino acid sequence reported in SEQ ID NQ:20; b) the amino acid sequence of HCDR2 is the amino acid sequence reported in SEQ ID NO: 21; c) the amino acid sequence of HCDR3 is the amino acid sequence reported in SEQ ID NO: 22; d) the amino acid sequence of LCDR1 is the amino acid sequence reported in SEQ ID NO: 9; e) the amino acid sequence of LCDR2 is the amino acid sequence YTS f) the amino acid sequence of LCDR3 is the amino acid sequence reported in SEQ ID NQ:10

[0062] The present invention further provides an isolated monoclonal antibody of the IgGl class or an antigen-binding fragment thereof comprising a variable domain of the light chainof SEQ ID NO: 8 and a variable domain of the heavy chain of SEQ ID NO: 19, wherein said variable domain of the light chain comprises an LCDR1 light chain complementarity determining region, an LCDR2 light chain complementarity determining region and an LCDR3 light chain complementarity determining region and wherein said variable domain of the heavy chain comprises an HCDR1 heavy chain complementarity determining region, an HCDR2 heavy chain complementarity determining region and an HCDR3 heavy chain complementarity determining region; these regions have been determined with IMGT.

[0063] In one embodiment the monoclonal antibody according to the present invention comprises a light chain with an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8, or fragments thereof; included is an antibody comprising an amino acid sequence of the light chain that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical or similar to the sequence of SEQ ID NO: 8.

[0064] In one embodiment the monoclonal antibody according to the present comprises a heavy chain with an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, or fragments thereof; an antibody comprising a heavy chain amino acid sequence that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical or similar to the sequence of SEQ ID NO: 19 is included.

[0065] A further object of the invention is an isolated nucleic acid for the production of the anti-human CD94 antibody, in particular an isolated nucleic acid comprising the sequences of SEQ ID NOs. 1 and 11 is preferred.

[0066] The nucleic acid according to the present invention may also be a nucleic acid comprising sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence homology to the sequences of SEQ ID NO. 1 and 11.

[0067] The present invention overcomes the disadvantages that characterize currently existing adaptive NK cells expansion methods. First, the present method allows to efficiently expand NKG2C+NK cells without the need to introduce genetically modified cells into the culture system, thus making this method safer and suitable for use in large-scale clinical applications. Furthermore, the present invention improves upon previous methods because it promotes the expansion of adaptive NK cells with high cytotoxic potential without inducingor significantly increasing the expression of inhibitory receptors such as the PD-1 receptor that could decrease the cytotoxic capabilities of expanded adaptive NK lymphocytes.

[0068] The present invention represents an alternative strategy to effectively promote the selective expansion of the population of adaptive NK cells NKG2C+(identified according to the selection strategies reported in fig.2 a-c).

[0069] The inventors have also developed an in vitro method improved compared to the methodologies described in the prior art based on the use of samples isolated from CMV+subjects. The inventors have demonstrated how this methodology allows obtaining high numbers of lymphocytes with the specific phenotype CD56+CD16+NKG2C+CD57+NKG2A“ self KIR+FcERy_ / lowSyk_ / lowand functionally specialized, compared to the use of starting samples isolated from CMV" subjects. In samples from CMV" subjects it is not possible to expand high numbers of NKG2C+cells which also lack the specific phenotype indicated above. The population of adaptive NK cells NKG2C+is present with variable frequency in the peripheral blood of healthy (Fig. 2d) and pathological CMV seropositive (CMV+) individuals, as well as in the peripheral blood of patients undergoing hematopoietic stem cell transplantation for the treatment of acute leukemia who frequently present CMV infection / reactivation in the first weeks or months post-transplant [8-10], The inventors have demonstrated how NK cells isolated or enriched from these subjects represent suitable sources for expanding adaptive NK cells with the present invention (Fig. 2a-c). The inventors have in fact demonstrated how in CMV seronegative subjects NKG2C+NK cells are present with low frequency (Fig. 2d) and therefore these individuals do not represent suitable donors. In the adult population of Western countries, CMV seropositivity is quite high and equal to approximately 83%

[0025] , making suitable donors easily available.

[0070] In one embodiment the sample used is a fresh isolated sample but the method according to the invention is also effective with thawed samples, allowing to select suitable donors and create a biobank of mononuclear cells from which to draw easily and rapidly to begin the expansion.

[0071] The method according to the present invention provides for:1. the selection of samples isolated from healthy CMV+donors preferably characterized by a frequency of adaptive NK lymphocytes NKG2C+equal to or greater than 20%;2. the enrichment of NK lymphocytes in preparations of mononuclear cells, obtained from peripheral blood by depletion of T lymphocytes preferably with anti-CD3 magnetic microbeads (e.g. MACSmicrobeads anti-CD3 Miltenyi Biotec) or by cell selection (sorting) by flow cytometry;3. the culture of the mononuclear cells selected after depletion of CD3+T lymphocytes for a period preferably between 10 and 14 days according to the protocol exemplified in fig. 3. The method according to the invention involves the use in the culture medium of at least one of the cytokines IL-2 and IL-15 in combination with the monoclonal antibody according to the invention (indicated by the English acronym mAb, monoclonal antibody). The monoclonal antibody according to the invention is used in a concentration between 0.0125 pg / ml and 2 pg / ml, preferably 0.125 pg / ml and is added at time 0 of the culture. In a preferred embodiment the antibody is removed from the culture on the fifth day.

[0072] The cytokines used are IL-2 and IL-15 in a concentration respectively between 50 lU / ml and 600 lU / ml, preferably 300 lU / ml, and 1 ng / ml and 40 ng / ml preferably 20 ng / ml. In a preferred embodiment IL-15 is used at a concentration of 20 ng / ml.

[0073] Mononuclear cells obtained from peripheral blood after depletion of CD3+T lymphocytes are represented by discrete percentages of NK lymphocytes (median=32%; range 25-52%), B lymphocytes (median=22%; range 10-34%) and monocytes (median=41%; range 20-60%). Following the application of the protocol, NK lymphocytes will be obtained almost exclusively, as indicated by the frequency of NK lymphocytes measured on days 10 and 14 post-culture both in the presence of IL-2 and IL-15 (median value NK=95% day 14 in IL-2+mAb, =96% day 14 in IL-15+mAb) (fig. 4).

[0074] The selective expansion of adaptive NK lymphocytes requires the application of a protocol consisting of an initial enrichment in NK lymphocytes, the culture of the cells withadequate doses of the recombinant human cytokine IL-2 or the recombinant human cytokine IL-15 and the use of a monoclonal antibody that must be added only at the beginning of the culture itself (fig. 3). The cytokines used (IL-2 and IL-15) are known to promote the survival, activation and proliferation of NK cells

[0026] and are commonly used in immunotherapeutic protocols

[0027] ,

[0075] The present invention allows the efficient expansion of NKG2C+NK lymphocytes in a relatively rapid manner, by means of a culture that provides a time preferably between 10 and 14 days as shown by the growth curves in fig. 5. In one embodiment the culture is maintained for 14 days but the person skilled in the art can establish the optimal duration of the culture. The use of IL-15 makes the method used more efficient allowing the recovery of a higher number of adaptive NK lymphocytes (fig. 5 right panel).

[0076] Selective expansion of adaptive NK lymphocytes is achieved by adding at the beginning of the culture (day 0) the monoclonal antibody according to the invention that recognizes the CD94 / NKG2C receptor complex expressed by adaptive NK cells. The culture performed in the absence of the antibody does not determine expansion of NKG2C+NK lymphocytes (fig. 5). This is also clearly demonstrated by the progressive dilution of the CFSE dye, with which the cells were labeled before the culture

[0014] , at the various times examined (fig. 6), comparing the proliferation and the progressive accumulation of NKG2C+NK lymphocytes in the presence of the mAb compared to the cytokine alone (Fig. 6, IL-2 panel b, IL-15 panel c).

[0077] The antibody used in the present invention was isolated and purified from the supernatant culture medium of a murine hybridoma generated using splenocytes obtained from immunocompetent mice (BALB / c strain) immunized with human NK cells and fusing them in vitro, thanks to the use of polyethylene glycol, with cells of a particular non-antibody- secreting murine myeloma line called P3U1, in order to obtain immortalized hybridomas

[0024] , The variable regions of the antibody reported above, and which include the CDR (Complementarity Determining Regions) portions, are able to interact with the antigen, i.e. the CD94 molecule, in a specific manner.

[0078] The NKG2C+NK lymphocytes that expand with the present invention are largely characterized by the expression of the terminal differentiation marker CD57. These cells prevail over the NK NKG2C+CD57- population which is scarcely present among the NK cellsisolated from peripheral blood on day 0 (not activated) and which, although increasing after 14 days of culture, represent a limited fraction of the expanded cells (fig. 6 and fig. 7).

[0079] The antibody employed in the present method interacts specifically with the CD94 molecule forming CD94 / NKG2x heterodimers thereby reacting with both the activating heterodimer CD94 / NKG2C and its inhibitory counterpart CD94 / NKG2A [28-31], The present method thus prevents the activation and expansion of non-adaptive CD94 / NKG2A+NK cells or NK cells co-expressing CD94 / NKG2A and CD94 / NKG2C, while allowing the selective activation and expansion of adaptive NK cell populations expressing CD94 / NKG2C and lacking CD94 / NKG2A (Fig. 8ab). This feature is advantageous since NKG2A represents a critical inhibitory checkpoint regulating NK cell cytotoxicity and its co-expression with NKG2C could decrease the therapeutic potential of NK cells. It should be noted that in the present method no expansion of NKG2C+NKG2A+NK cells is observed. This is associated with the prevalence of the inhibitory signal generated by the stimulation of the CD94 / NKG2A heterodimer over the activating signal due to the CD94 / NKG2C heterodimer when they are simultaneously stimulated by the anti-CD94 monoclonal antibody according to the invention (fig. 8ab)

[0032] , Any increased expression of NKG2A on NKG2C+NK lymphocytes due to exposure to cytokines, as described in patent EP3539552A1, therefore does not occur in the present system, providing a further advantage to this method.

[0080] As previously mentioned, adaptive NK lymphocytes generated following cytomegalic infection are characterized by the expression of inhibitory KIR receptors of the "self" type, that is, they recognize HLA class I molecules expressed by healthy cells of the individual.

[0081] NKG2C+NK cells expanded according to the present method are characterized not only by the absence of NKG2A, but also by the presence of a single self-type KIR receptor (usually KIR2DL2-L3 or KIR2DL1, rarely KIR3DL1 as expected from the literature [10, 12, 13]) that was already present on the pre-expansion cells (fig. 8 c, d, e). These self-KI R+NK cells express high levels of the cytotoxic molecules perforin and granzyme B both pre- and post-expansion (not shown). The almost exclusive expression of a single inhibitory receptor KIR opens the possibility of exploiting the cytotoxic capabilities of adaptive NK lymphocytes expanded with the present method against tumors such as acute leukemias that express HLA-L For this purpose, healthy CMV+donors must be selected whose expanded NKG2C+NKG2A“ single self- KIR+NK lymphocytes are characterized by a KIR receptor that recognizes an HLA-I moleculeabsent in the genotype (and therefore not expressed) of the patient affected by leukemia. The expanded adaptive NK lymphocytes will be alloreactive towards the leukemic cells of the patient to be treated, i.e. their cytotoxic activity will not be blocked by KIR-HLA interactions that normally regulate the cytolytic activity of NK cells [33, 34],

[0082] It is important to note that following mAb-mediated expansion, some peculiar molecular characteristics of NKG2C+adaptive NK cells are also maintained. In particular, a variable fraction of adaptive NK lymphocytes isolated from peripheral blood of CMV+subjects lack the expression of the adaptor proteins FceRy and Syk following epigenetic modifications induced by cytomegalic infection [12-14] (fig. 9a day 0). These modifications are preserved post-expansion, as demonstrated by the frequency of NKG2C+CD57+FceRy- and Syk- NK lymphocytes on day 14 (fig. 9a day 0 vs 14). The adaptor molecule CD3^ is instead normally expressed, as is the activating receptor CD16 (fig. 9a), a receptor for the Fc fraction of IgG that mediates the ADCC (antibody-dependent cellular cytotoxicity) mechanism. As previously described, the lower expression of FceRy is associated with marked ADCC capabilities of adaptive NK lymphocytes that will then be found also in expanded adaptive NK lymphocytes (see fig. 9).

[0083] The cytotoxic activity of NK cells can be dampened by the presence of inhibitory receptors called immune checkpoints whose expression can be induced or increased following particular stimuli (e.g. PD-1, TIM-3, LAG-3, TIG IT) [35-37], For instance, NK lymphocytes from patients affected by various types of tumors can express the inhibitory receptor PD-1 that decreases their cytolytic capabilities towards tumor cells expressing PD-1 ligands (PD-L1 / 2)

[0037] , It is possible that soluble factors present in the tumor microenvironment or cell-cell interactions favor the expression of PD-1 on NK cells. Furthermore, PD-1 and LAG-3 expression was also induced in vitro in NK lymphocytes stimulated for 7 days by monoclonal antibodies against the NKG2C receptor in the presence of IL-15. These NKG2C+PD-1+NK cells showed signs of senescence and functional exhaustion

[0038] , In line with this data, an increased expression of PD-1 was also clearly described in the expansion method of NKG2C+NK lymphocytes indicated in the patent EP3539552A1 and in the study by Haroun-lzquierdo et al.

[0022] , Unlike previous methods, the expression of the inhibitory receptor PD-1 was never observed at significant levels in NKG2C+NK lymphocytes obtained following the expansionmediated by the monoclonal antibody used in the present invention (fig. 9b). This feature represents a significant advantage compared to the previous methods mentioned above.

[0084] As regards the other immune checkpoints analyzed in this method, it is important to underline that the expression of the TIGIT receptor, which is already observable on day 0 on adaptive lymphocytes, remains unchanged on day 14 post-expansion. Finally, the expression of the TIM-3 and LAG-3 checkpoints, substantially absent on day 0, is induced by exposure to cytokines, both IL-2 and IL-15, even in the absence of the stimulus provided by the mAb. The mAb addition does not significantly increase the expression of TIM-3 and LAG-3 compared to the cytokine alone (fig. 9b).

[0085] The adaptive NK lymphocytes NKG2C+CD57+NKG2A’ selfKIR+selectively expanded according to the present invention were found to be highly functional in degranulation assays (CD107a assay) against lines expressing HLA-E (221E), ligand of the CD94 / NKG2C receptor. Furthermore, such expanded lymphocytes show a high capacity to respond via ADCC to tumor cells opsonized with IgG, as indicated by the high levels of degranulation in response to the lymphoma cell line Raji, coated with the monoclonal antibody Rituximab (anti-CD20) (fig. 10).

[0086] The high functional capacity of the adaptive NK lymphocytes expanded with the present method demonstrates that this invention can be used to design immunotherapies that combine the administration to patients of the cells expanded in vitro together with monoclonal antibodies directed against tumor-associated antigens (e.g. CD20, EGFR, HER-2) or innovative molecules such as bispecific or trispecific antibodies or engagers [21, 39], These molecules recognize and bind antigens expressed on tumor cells and simultaneously stimulate via CD16 the cytotoxic capabilities of the adaptive NK previously expanded in vitro, thus directing their potent cytotoxic activity against the tumor target. Alternatively, the adaptive NK cells can be expanded directly in vivo by administering to patients the mAb or its derivatives further modified to stimulate both the expansion of the adaptive NK cells and their anti-tumor cytotoxic capabilities.

[0087] In fact, the present invention also proposes the use of the antibody, or fragments thereof or its derivatives as immunological tools to be used not only ex vivo, but also in vivo to expand specific populations of NK cells for therapeutic purposes in patients affected by haematological pathologies and solid tumours or immunodeficiencies.

[0088] It is therefore an object of the present invention to provide a method forthe treatment of a patient affected by acute myeloid or lymphoid leukemia, lymphomas, myeloma, ovarian, peritoneal, gastric, colorectal, breast, head and neck, pancreatic carcinoma, sarcomas and / or brain tumors, wherein said method includes a first phase, in vitro, to expand adaptive NK lymphocytes NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / Iow, such phase comprising the steps of: i. i. Isolating from a biological sample of a subject with CMV+serology, a population of peripheral blood mononuclear cells, ii. Performing a depletion of T lymphocytes to obtain a population enriched with NK lymphocytes, iii. Cultivating the population obtained at point ii. in a culture medium comprising the anti CD94 antibody according to the invention and at least one of the cytokines IL-2 or IL- 15 for a time between 10 and 14 days, iv. obtaining an expanded population of NK cells enriched with NKG2C+CD57+NKG2A“ selfKI R+FcERy- / l0Wand Syk- / Iowcells; and a second phase wherein the population obtained in phase iv.) is used for the treatment of said patients.

[0089] In a preferred embodiment, in the first phase the isolated sample of the subject with CMV+serology presents NK cells expressing KIR specific for HLA-C1 or for HLA-C2 and in step iv.) an expanded population of NK cells enriched by NKG2C+CD57+NKG2A“selfKIR2DL2-L3+FC£RY- / |OWand Syk- / Iowor NKG2C+CD57+NKG2A~ selfl<IR2DLl+FcsRy- / lowand Syk- / Iowcells is obtained.

[0090] In particular, the method described here may be used to develop the following immunotherapeutic strategies:-infusion of adaptive NK lymphocytes expanded ex vivo according to the method described here in patients affected by tumors of various histological types such as acute myeloid leukemia, ovarian, peritoneal, gastric, colorectal, breast, head and neck carcinoma for which some clinical trials are already underway with adaptive NK lymphocytes expanded with other methods (e.g. NCT03081780, NCT03213964, NCT03319459).-engineering of antibody fragments, in particular of the CDR portions or parts therefrom, in order to generate new molecules derived from the original mAb, but more efficient andflexible for in vivo use. For instance, thanks to technological platforms already used in molecular biology laboratories and by companies in the biotechnology sector [21, 39] it will be possible to generate mAb-derived molecules containing functional portions of the cytokine (e.g. IL-15) necessary to support the proliferation of NK lymphocytes. A molecule of this type can be used for the ex vivo expansion of adaptive NK lymphocytes and can also be used to obtain the proliferation and activation of adaptive NK lymphocytes in vivo, thus avoiding the complex preparation of cells to be infused according to GMP criteria. Fragments or derivatives of the mAb could be used to generate bispecific or trispecific antibodies containing the CDR portions or parts therefrom, combined with mAbs or derivatives therefrom specific for tumor antigens together with functional portions of cytokines. Such molecules can activate the effector functions of adaptive NK lymphocytes against tumor cells while supporting the proliferation of the adaptive NK population.- generation of CAR-modified NK [21, 40] starting from expanded adaptive NK lymphocytes, therefore endowed with high longevity and functionality, to be used in precision medicine against haematological and solid tumours, capable of recognising specific antigens on tumour cells through the CAR molecule.

[0091] A further element that potentially makes this method more advantageous and flexible is given by the possibility of using both IL-2 and IL-15 in the culture system. Depending on the type of immunotherapy to be applied, itwill be possible to evaluate which of the two cytokines is more suitable to limit the adverse effects that have been associated with them, such as the generation of regulatory T lymphocytes induced by IL-2

[0041] ,

[0092] Finally, one of the most relevant advantages provided by this method is that of efficiently expanding NKG2C+NK lymphocytes without the need to introduce genetically modified cells into the culture system as required by previous methods described in WO2014037422A1 and in the related publication

[0022] , This method is therefore safer and more suitable for use in large-scale clinical applications.

[0093] In this regard, it is important to specify that, if necessary, the monoclonal antibody can be eliminated from the culture after 5 days from seeding of the cells, without affecting either the expansion of the adaptive NK lymphocytes or their phenotypic and functional characteristics.

[0094] The indicated system can therefore be further adapted and improved to meet the needs of large-scale production and possible engineering of the expanding cells.Description of materials and methodsBiological samples used

[0095] Peripheral blood samples were provided by the Transfusion Center-IRCCS Ospedale Policlinico San Martino (Genoa, Italy) and collected from healthy adult donors. Donors gave their informed consent to participate in this study in accordance with the Declaration of Helsinki. The use of these samples was approved bythe Ethics Committee of the Liguria Region (Protocol No. 39 / 2012, CER Liguria number: DB ig 10125).Peripheral blood sample processing

[0096] Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation (Ficoll-Hypaque solution, Sigma-Aldrich, St. Louis, MO), analyzed by immunofluorescence and flow cytometry (see next section) to assess donor eligibility, and frozen. Subsequently, aliquots of mononuclear cells from selected donors were thawed and processed as indicated.

[0097]

[0097] CMV serology of the donors used was assessed on plasma obtained from peripheral blood, using an enzyme immunoassay forvirus-specific IgG immunoglobulins (CMV IgG ELISA Kit, Technogenetics, Lodi, Italy).Anti-CD94 monoclonal antibody

[0098]

[0098] The monoclonal antibody used in the present invention was produced in our laboratory and purified with techniques previously described [24, 28-31], Briefly, hybridomas were generated by immunizing immunocompetent mice of the BALB / c strain (code 028 BALB / c, supplied by Charles River laboratories Italia S.r.l. Lecco) with human NK cells, isolated from the peripheral blood of healthy adult donors using specific kits according to the supplier's instructions (NK cell isolation Kit Mlltenyi Biotec, Bergisch Gladbach, Germany). The splenocytes obtained from the immunized mice were subjected to an in vitro fusion process, thanks to the use of polyethylene glycol, with cells of a particular non-antibody-secreting murine myeloma line called P3U1 (P3X63AgU.l, ATCC n° CRL-1597), in order to obtain immortalized hybridomas. Subsequently, the antibody in question was selected using specific assays. The antibody is of murine origin, of the IgGl isotype and has been validated for itsspecificity. The antibody recognizes the CD94 / NKG2C and CD94 / NKG2A heterodimeric complexes on human cells, interacting specifically with the CD94 molecule. The variable regions of the antibody that include the CDR (Complementarity Determining Regions) portions have been sequenced and are reported in the sequence listing.Preparation of mononuclear cells, media and antibody used for culture

[0099] Mononuclear cells obtained from peripheral blood were subjected to T lymphocyte depletion by positive selection with anti-CD3 magnetic microbeads (MACSmicrobeads Miltenyi Biotec, Bergisch Gladbach, Germany). The preparation of mononuclear cells deprived of T lymphocytes and enriched in NK lymphocytes was cultured according to the process schematized in figure 3. Initially, CD3+T lymphocytes were depleted from peripheral blood mononuclear cells of previously selected healthy CMV+donors, from which NK lymphocytes, B lymphocytes and monocytes were obtained. Post-depletion mononuclear cells are cultured with IL-2 or IL-15 at final concentrations of 50 lU / ml to 600 lU / ml and 1 ng / ml to 40 ng / ml, respectively, in the examples in the figure IL-2 300 lU / ml and IL-15 20ng / ml, in the presence or absence of the monoclonal antibody (mAb) at a concentration of 0.0125 pg / ml to 5 pg / ml (the mAb is added only on day 0), in the examples in the figure at a final concentration of 0.125 pg / ml. The cells were seeded at a concentration of approximately 5xl05 / ml in 96-well plates. On day 5, fresh medium with the corresponding cytokine was added, while on days 7 and 10 a 40%-50% medium change was performed, always with the relevant cytokine. During the culture, it was necessary to regularly add fresh medium containing the corresponding cytokine. On days 5, 7, 10 and 14, a portion of the cultured cells was counted and characterized by flow cytometric analysis. Finally, on day 14 the cells were recovered and used for further phenotypic and functional studies.

[0100]

[0100] The medium used in the cultures was RPMI-1640, supplemented with 2 mM L- glutamine, a mixture of penicillin-streptomycin (1%), heat-inactivated fetal bovine serum (10%), and supplemented with IL-2 (Proleukin, Chiron, Emeryville, CA), or IL-15 (Peprotech, London, UK) at the concentrations indicated above.

[0101]

[0101] Cells were seeded at a concentration of 5xl05 / ml in 96-well plates (105cells post-depletion per well) in the presence or absence of the monoclonal antibody (mAb) at a final concentration of 0.125 pg / ml. The mAb was added only on day 0. On day 5, fresh medium with the corresponding cytokine was added, while on days 7 and 10 a 40%-50% mediumchange was performed, always with the relevant cytokine. On the indicated days, cells were collected, counted and subjected to the necessary flow cytometric analyses to characterize their phenotype and function as described in the text.Proliferation assay

[0102] In each experiment, a portion of the post-CD3 lymphocyte depletion mononuclearcells was labeled with the intracellular fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Lifetechnologies, Oregon, USA). Briefly, cells were thoroughly washed, resuspended in medium at a concentration of 107 / ml, and incubated for 10 minutes at 37°C with a CFSE solution (2.5 pM). After two washes with complete medium (with serum added), the CFSE-labeled cells were cultured according to the protocol shown in figure 3 and described in detail in the previous section. On the indicated days, cells were subjected to flow cytometric investigations to evaluate their proliferative capacity indicated by the dilution of the CSFE dye.Monoclonal antibodies and cytofluorimetric analyses

[0103] The different monoclonal antibodies used in flow cytometric analyses are reported in Table 1 where the clone of the antibody and the company that supplied it are also indicated. The isotype-specific secondary reagents (goat anti-mouse) used are also indicated in Table 1. When mAbs labeled with fluorochromes were used, appropriate isotype controls supplied by the same companies were used. In immunofluorescence experiments, cells were incubated with the appropriate antibody mixtures for 30 minutes at 4°C and washed with PBS supplemented with FCS (5%).

[0104] NK cell phenotype and effector functions were analyzed by selecting NK cells by physical parameters and by the combined use of anti-CD56, anti-CD16, anti-CD3, anti-CD19, anti-CD14 mAbs as described in the selection strategies shown in fig. 2a-c. To evaluate the different NKG2C / CD57 / NKG2A / KIR subpopulations, the appropriate mAb mixtures reported in Table 1 were used. For intracellular flow cytometric analyses, cells were fixed and permeabilized with the Foxp3 permeabilization kit (Miltenyi Biotec) according to the manufacturer's instructions. Before proceeding with sample acquisition, the Fixable Viability Stain reagent (BD Biosciences) was added in order to exclude dead cells from subsequent analyses.

[0105] Flow cytometric analyses were performed on an eight-color BD FACSVerse (Becton Dickinson, Mountain View, CA) and data were analyzed by FACSuite software version 1.0.6.Functional degranulation assays

[0106] In order to evaluate the cytotoxic capacity of expanded NK cells, the degranulation test was used. This test represents a valid alternative to the assays that evaluate the cytotoxic capacity of NK lymphocytes by directly measuring the death of the target cells with which the NK lymphocytes are placed in contact. In particular, the degranulation test indirectly measures the cytotoxic capacity of NK cells since it is based on the quantification of NK cells that, following contact with the target cell (e.g. tumor cell line), release the contents of their cytotoxic granules close to the target cell itself by exocytosis. These granules contain perforin and granzymes, molecules responsible for killing the target cell. Following the release of the cytotoxic granules, i.e. degranulation, NK cells express the molecule CD107a, a transmembrane protein, a marker of cytotoxic granules on the plasma membrane. Therefore, NK lymphocytes that have undergone degranulation and killed the target are easily identifiable and quantifiable by flow cytometric analysis. It is sufficient that NK lymphocytes are held in contact with the target cell in the presence of an anti-CD107a antibody to identify CD107a+NK cells as those NK cells capable of killing the target that have released perforin and granzymes against it.

[0042]

[0107] On day 14 of culture, cytokine+mAb expanded cells were harvested, washed and incubated with different tumor cell lines at a 1:1 effector NK cell: target cell ratio for 3 hours in culture medium supplemented with anti-CD107a-PE mAb. In particular, the following tumor cell lines were used: K562 (ATCC n° CCL-243), LCL 721.221wild type (ATCC n° CRL-1855, 221wt), LCL721.221AEH [221AEH, LCL 721.221 transfected to express HLA-E, kind gift of prof. Miguel Lopez-Botet and Dr. Aura Muntasell (Hospital del Mar Medical Research Institute IMIM, Barcelona, Spain; Department of Medicine and Life Sciences, Univ. Pompeu Fabra, Barcelona, Spain), originally developed by Geraghty D. et al

[0043] (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA)], RAJI, (ATCC n°CCL-86), RAJI previously opsonized with the mAb anti-CD20 Rituximab (1 pg / ml). Basal degranulation of NK cells was assessed in 3-hour cultures without tumor cell lines.

[0108] Subsequently, cells were collected and surface-labeled with the mAb mixtures necessary for the identification and characterization of NK cells, i.e., anti-CD56-PC7, anti-CD3-Viogreen, anti-CD19-Viogreen, anti-CD14-Viogreen, anti-CD16-PercpCy5.5, anti-CD57- VioBlue, anti-NKG2C followed by appropriate secondary reagent anti-mouse lgG2b conjugated to APC or APC-CY7, for 30 min at 4°C. Cells were then stained with Fixable Viability Stain (BD Biosciences), washed, and analyzed by flow cytometry.Statistical analyses

[0109] Non-parametric Wilcoxon-Mann-Whitney tests were used. Statistical significance (p- value) is indicated in the different graphs (*p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001). Graphical representations and statistical analyses were performed with GraphPad Prism 8 (GraphPad Software, La Jolla, CA).References1. Vivier, E., et al., Functions of natural killer cells. Nat Immunol, 2008. 9(5): p. 503-10.2. Moretta, A., et al., Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol, 2001. 19: p. 197-223.3. Parham, P., MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol, 2005. 5(3): p. 201-14.4. Ivarsson, M.A., J. Michaelsson, and C. Fauriat, Activating killer cell Ig-like receptors in health and disease. Front Immunol, 2014. 5: p. 184.5. Braud, V.M., et al., HLA-E binds to natural killer cell receptors CD94 / NKG2A, B and C. Nature, 1998. 391(6669): p. 795-9.6. Elliott, J.M. and W.M. Yokoyama, Unifying concepts of MHC-dependent natural killer cell education. Trends Immunol, 2011. 32(8): p. 364-72.7. Guma, M., et al., Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood, 2004. 104(12): p. 3664-71.8. Foley, B., et al., Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood, 2011.9. Della Chiesa, M., et al., Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus? Blood, 2012. 119(2): p. 399-410.10. Muccio, L., et al., Analysis of memory-like natural killer cells in human cytomegalovirus- infected children undergoing alphabeta+T and B cell-depleted hematopoietic stem cell transplantation for hematological malignancies. Haematologica, 2016. 101(3): p. 371-81.11. Lopez-Botet, M., et al., Adaptive NK cell response to human cytomegalovirus: Facts and open issues. Semin Immunol, 2023. 65: p. 101706.12. Schlums, H., et al., Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function. Immunity, 2015. 42(3): p. 443-56.13. Lee, J., et al., Epigenetic Modification and Antibody-Dependent Expansion of Memorylike NK Cells in Human Cytomegalovirus-Infected Individuals. Immunity, 2015. 42(3): p. 431- 42.14. Muccio, L., et al., Late Development of FcERy(neg) Adaptive Natural Killer Cells Upon Human Cytomegalovirus Reactivation in Umbilical Cord Blood Transplantation Recipients. Front Immunol, 2018. 9: p. 1050.15. Costa-Garcia, M., et al., Antibody-Mediated Response of NKG2Cbright NK Cells against Human Cytomegalovirus. J Immunol, 2015. 194(6): p. 2715-24.16. Russo, A., et al., NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: dynamics and clinical implications. Blood, 2018. 131(2): p. 247-262.17. Cichocki, F., et al., CD56dimCD57+NKG2C+ NK cell expansion is associated with reduced leukemia relapse after reduced intensity HCT. Leukemia, 2016. 30(2): p. 456-63.18. Cichocki, F., et al., Adaptive NK cell reconstitution is associated with better clinical outcomes. JCI Insight, 2019. 4(2).19. Yu, X.X., et al., Donor NKG2C homozygosity contributes to CMV clearance after haploidentical transplantation. JCI Insight, 2022. 7(3).20. Myers, J. A. and J.S. Miller, Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol, 2021. 18(2): p. 85-100.21. Shin, E., et al., Understanding NK cell biology for harnessing NK cell therapies: targeting cancer and beyond. Front Immunol, 2023. 14: p. 1192907.22. Haroun-lzquierdo, A., et al., Adaptive single-KIR(+)NKG2C(+) NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA- mismatched acute myeloid leukemia. J Immunother Cancer, 2022. 10(11).23. Hammer, Q„ et al., Peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells. Nat Immunol, 2018. 19(5): p. 453-463.24. Kohler, G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975. 256(5517): p. 495-7.25. Diaz-Decaro, J., et al., A systematic literature review of the economic and healthcare resource burden of cytomegalovirus. Curr Med Res Opin, 2023. 39(7): p. 973-986.26. Cooper, M.A., T.A. Fehniger, and M.A. Caligiuri, The biology of human natural killer-cell subsets. Trends Immunol, 2001. 22(11): p. 633-40.27. Lui, G., et al., Exploiting an Interleukin-15 Heterodimeric Agonist (N803) for Effective Immunotherapy of Solid Malignancies. Cells, 2023. 12(12).28. Moretta, A., et al., Human natural killer cell receptors for HLA-class I molecules. Evidence that the Kp43 (CD94) molecule functions as receptor for HLA-B alleles. J Exp Med, 1994. 180(2): p. 545-55.29. Pende, D., et al., HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex. Eur J Immunol, 1997. 27(8): p. 1875-80.30. Cantoni, C., et al., The activating form of CD94 receptor complex: CD94 covalently associates with the Kp39 protein that represents the product of the NKG2-C gene. Eur J Immunol, 1998. 28(1): p. 327-38.31. Della Chiesa, M., et al., Human Cytomegalovirus Infection Promotes Rapid Maturation of NK Cells Expressing Activating Killer Ig-Like Receptor in Patients Transplanted with NKG2C- / - Umbilical Cord Blood. J Immunol, 2014.32. Brostjan, C., et al., Differential expression of inhibitory and activating CD94 / NKG2 receptors on NK cell clones. J Immunol Methods, 2002. 264(1-2): p. 109-19.33. Locatelli, F., et al., NK Cells Mediate a Crucial Graft-versus-Leukemia Effect in Haploidentical-HSCT to Cure High-Risk Acute Leukemia. Trends Immunol, 2018. 39(7): p. 577- 590.34. Dhuyser, A., et al., KIR in Allogeneic Hematopoietic Stem Cell Transplantation: Need for a Unified Paradigm for Donor Selection. Front Immunol, 2022. 13: p. 821533.35. Khan, M., S. Arooj, and H. Wang, NK Cell-Based Immune Checkpoint Inhibition. Front Immunol, 2020. 11: p. 167.36. Sivori, S., et al., Inhibitory Receptors and Checkpoints in Human NK Cells, Implications for the Immunotherapy of Cancer. Front Immunol, 2020. 11: p. 2156.37. Pesce, S., et al., Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization. J Allergy Clin Immunol, 2017. 139(1): p. 335-346.e3.38. Merino, A., et al., Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming. J Clin Invest, 2019. 129(9): p. 3770-3785.39. Della Chiesa, M., et al., NK Cell-Based Immunotherapy in Colorectal Cancer. Vaccines (Basel), 2022. 10(7).40. Liu, E., et al., Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia, 2018. 32(2): p. 520-531.41. Abbas, A.K., et al., Revisiting IL-2: Biology and therapeutic prospects. Sci Immunol, 2018. 3(25).42. Alter, G., J.M. Malenfant, and M. Altfeld, CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods, 2004. 294(1-2): p. 15-22.43. Lee, N., et al., HLA-E is a major ligand for the natural killer inhibitory receptor CD94 / NKG2A. Proc Natl Acad Sci U S A, 1998. 95(9): p. 5199-204.

Claims

Claims1. A monoclonal antibody isolated against human CD94 or an antigen-binding fragment thereof comprising three heavy chain complementarity determining regions, HCDR1, HCDR2, HCDR3 and three light chain complementarity determining regions LCDR1, LCDR2, LCDR3 wherein: a) the amino acid sequence of HCDR1 is the amino acid sequence reported in SEQ ID NO:20; b) the amino acid sequence of HCDR2 is the amino acid sequence reported in SEQ ID NO: 21; c) the amino acid sequence of HCDR3 is the amino acid sequence reported in SEQ ID NO: 22; d) the amino acid sequence of LCDR1 is the amino acid sequence reported in SEQ ID NO: 9; e) the amino acid sequence of LCDR2 is the amino acid sequence YTS f) the amino acid sequence of LCDR3 is the amino acid sequence reported in SEQ ID NQ:

102. An isolated monoclonal antibody of the IgGl class or an antigen-binding fragment thereof comprising a light chain variable domain of SEQ ID NO: 8 and a heavy chain variable domain of SEQ ID NO: 19, wherein said light chain variable domain comprises an LCDR1 light chain complementarity determining region, an LCDR2 light chain complementarity determining region and an LCDR3 light chain complementarity determining region and wherein said heavy chain variable domain comprises an HCDR1 heavy chain complementarity determining region, an HCDR2 heavy chain complementarity determining region and an HCDR3 heavy chain complementarity determining region, determined by IMGT.

3. An isolated IgGl class monoclonal antibody or an antigen-binding fragment thereof according to the preceding claim wherein the antibody binds human CD94.

4. A monoclonal antibody or a fragment thereof according to anyone of claims 1 to 3 wherein the LCDR1 region comprises amino acids 9 to 14 of SEQ ID NO: 8 and wherein the LCDR2 regioncomprises amino acids 32 to 34 of SEQ ID NO: 8 and wherein the LCDR3 region comprises amino acids 71 to 79 of SEQ ID NO:

85. A monoclonal antibody or a fragment thereof according to anyone of claims 1 to 4 wherein the HCDR1 region comprises amino acids 8 to 15 of SEQ ID NO: 19 and wherein the HCDR2 region comprises amino acids 33 to 40 of SEQ ID NO: 19 and wherein the HCDR3 region comprises amino acids 79 to 89 of SEQ ID NO:

196. A monoclonal antibody or a fragment thereof according to anyone of claims 1 to 5 for use in medicine7. Use of an isolated nucleic acid, encoding the monoclonal antibody according to anyone of claims 1 to 6 for the production of an anti-human CD94 antibody8. An isolated nucleic acid according to the preceding claim comprising the sequences of SEQ ID NO. 1 and 11.

9. An in vitro method to expand adaptive NK lymphocytes NKG2C+CD57+NKG2A“ selfKIR+FCERV“ / IOWand Syk“ / Iowcomprising: i. Isolating from a biological sample of a subject with CMV+serology, a population of peripheral blood mononuclear cells, ii. Performing a depletion of T lymphocytes to obtain a population enriched in NK lymphocytes, iii. Cultivating the population obtained in point ii. in a culture medium comprising the anti-CD94 antibody according to anyone of claims 1 to 5 and at least one of the cytokines IL-2 or IL-15 for a time of 10 to 14 days, wherein said method produces an expanded population of NK lymphocytes enriched with NKG2C+CD57+NKG2A“selfKIR+Fc£Ry- / lowand Syk- / Iowcells.

10. The method according to the preceding claim wherein the NKG2C+CD57+NKG2A“selfKIR+Fc£Ry_ / lowand Syk_ / Iowcell population is further characterized by the absence of the inhibitory receptor PD-111. The method according to anyone of claims 9 and 10 wherein the frequency of NKG2C+adaptive NK lymphocytes in the sample at point i. is equal to or greater than 20%12. The method according to anyone of claims 9-11 wherein the isolated sample of the subject with CMV+serology presents NK lymphocytes expressing KIRs specific for HLA-C1 or for HLA- C2 and wherein said method produces an expanded population of NK lymphocytes enriched with NKG2C+CD57+NKG2A“selfKIR2DL2-L3+Fc£Ry- / lowand Sylc / Iowcells or NKG2C+CD57+NKG2A“selfKIR2DLl+Fc£Ry- / low andSylc / Iowcells.

13. The method according to anyone of claims 9-12 wherein on day 5 of culture, culture medium comprising at least one of the cytokines IL-2 or IL-15 is added and on days 7 and 10 a change of medium is carried out wherein an amount of medium ranging from 40% or 50% of the medium of said culture is substituted14. The method according to anyone of claims 9-13 wherein the antibody is removed on day 5 of the culture15. The method according to anyone of claims 9-14 wherein the monoclonal antibody against CD94 is used in a concentration of between 0.0125 pg / ml and 2 pg / ml, preferably 0.125 pg / ml16. The method according to anyone of claims 9-15 wherein the cytokine used is IL-2 in a concentration between 50 lU / ml and 600 lU / ml, preferably 300 lU / ml17. The method according to anyone of claims 9-16 wherein the cytokine used is IL-15 in a concentration between 1 ng / ml and 40 ng / ml, preferably 20ng / ml18. A population of NK lymphocytes enriched with NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / Iowcells expanded with the method according to anyone of claims 9-17 for use in the treatment of haematological diseases, solid tumours, immunodeficiencies.

19. A population of NK lymphocytes enriched with NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / Iowcells expanded with the method according to anyone of claims 9-17 for use according to the preceding claim wherein the pathology is selected from acute myeloid and lymphoid leukemias, lymphomas, myeloma, ovarian, peritoneal, gastric, colorectal, breast, head and neck, pancreatic carcinomas, sarcomas and brain tumors.

20. A composition comprising adaptive NK lymphocytes NKG2C+CD57+NKG2A“selfKIR+FcsRy- / lowand Syk_ / Iowdirectly obtainable with the method according to anyone of claims 9-17.

21. Adaptive NK lymphocytes NKG2C+CD57+NKG2A- selfKIR+Fc£Ry- / lowand Syk- / IowPD-1" or composition comprising adaptive NK lymphocytes NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk / lowPD-1" for use in the treatment of hematological diseases, solid tumors, immunodeficiencies.

22. Adaptive NK lymphocytes NKG2C+CD57+NKG2A-selfKIR+Fc£Ry- / lowand Syk- / IowPD-1" or a composition comprising adaptive NK lymphocytes NKG2C+CD57+NKG2A“ selfKIR+Fc£Ry_ / lowand Syk_ / IowPD-1- for the use according to the preceding claim wherein the pathology is selected from acute myeloid and lymphoid leukemias, lymphomas, myeloma, ovarian carcinoma, peritoneal carcinoma, gastric carcinoma, colorectal carcinoma, breast carcinoma, head and neck carcinoma, pancreatic carcinoma, sarcomas and brain tumors.

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